Journal: Nature Communications
Article Title: NG2 glia-derived GABA release tunes inhibitory synapses and contributes to stress-induced anxiety
doi: 10.1038/s41467-021-25956-y
Figure Lengend Snippet: a Cartoon images illustrating the sniffer-patch method in co-cultured NG2 cells and HEK-293T cells. Scale bar, 20 μm. b Representative image showing primary cultured NG2 cells (in green) from Pdgfrα-creER TM ; ChR2-eYFP newborn mice brain with GABA A Rs-mCherry transfected HEK-293T cell (in red). Scale bar, 20 μm. c The representative trace shows an example of ChR2-evoked photocurrents by 100 ms blue light stimulation in a cultured NG2 cell. Bar graph shows −365.3 ± 25.1 pA peak photocurrent induced by blue light stimulation in cultured NG2 cells, n = 10 cells. d , e Representative traces ( d ) and summarized bar graph ( e ) show that 25 μM GABA application induces a distinct GABA A R-mediated current that is significantly abolished by GABA A R antagonist bicuculline in transfected HEK-293T cells. GABA A R-mediated currents: control: −526.2 ± 67.2 pA, n = 6 cells; in the presence of bicuculline: −32.9 ± 10.7 pA, n = 6 cells, two-tailed paired t test, P = 0.0005. f Representative images show robust [Ca 2+ ] i elevations in cultured NG2 cells loaded with the calcium indicator Rhod2-AM (5 µM) after 15 Hz 60 s blue light stimulation. Bar graph summary below shows the time course of blue light stimulation-induced [Ca 2+ ] i increase in cultured NG2 cells. n = 53 cells from 3 mice. Data are normalized by the mean fluorescence intensity obtained during the control period (0–30 s before the stimulation triggers) for each cell. g Representative traces and summarized bar graph show a significant increase of GABA A R-mediated tonic current in sniffer-patched transfected HEK-293T cell in the presence of blue light stimulation of cultured NG2 cells (middle panel) compared with its basal tonic inhibition in the absence of blue light stimulation (upper panel) obtained from a Pdgfrα-creER TM ;ChR2 mouse. This increased tonic GABA current is abolished by the cotransfection with exocytosis blocker TeTX in the presence of NG2 cells photoactivation (lower panel). Basal tonic GABA currents in transfected HEK cells without blue light stimuli: 6.57 ± 0.82 pA, n = 10 cells; tonic GABA currents in the presence of blue light stimuli: 28.27 ± 3.61 pA, n = 15 cells; tonic GABA currents in the presence of TeTX and blue light stimuli: 12.38 ± 2.33 pA, n = 12 cells. ANOVA Tukey–Kramer Multiple Comparisons, *** indicates P < 0.001. h Bar graph summary showing increased GABA concentrations in purified ChR2-expressing NG2 cells after blue light stimulation compared with its control with high-performance liquid chromatography (HPLC) analysis. n = 4 and 5 independent experiments for ChR2 + -NG2 cells and ChR2 - -NG2 cells, respectively. P values as indicated, two-tailed paired t test. i GABA levels in synaptosomes isolated from purified NG2 cells and GAD67-GFP interneurons with HPLC. Bar graph showing an average GABA concentration of 5.76 ± 1.04 ng/mg synaptosomes in NG2 glia (right panel). n = 5 tested samples. j The bar graph summary shows GABA contents in synaptosomes in both purified NG2 cells and GAD67+ interneurons compared with hippocampal tissue. n = 11 tested samples for purified primary cultured NG2 cells, n = 2 and 3 tested samples for GAD67 interneurons and hippocampi from 4 GAD67-GFP and 3 C57BL/6 mice, respectively. k The pie graph shows a component percentage of vesicle-associated membrane protein (VAMP)-encoded genes through RNA-sequencing data analysis of isolated NG2 glia in adult brain by FACS at postnatal 3–4 weeks. The representative image below shows a precise colocalization of transfected VAMP2-pHuji plasmid (in red) with anti-VAMP2 antibody (in green) in primary cultured NG2 cells. Scale bar, 20 μm. l Representative TIRFM images show the total VAMP2-pHuji laden vesicular fusion events at 0, 2.5, 5, 7.5, and 10 s with the image size of 10 × 10 μm 2 before (upper panel) and after (lower panel) blue light stimulation (10 Hz, 60 s) in the absence (control panels) or the presence of TeTX (TeTX panels), respectively. The average cumulative graphs of fusion events during a 10 s time window before and after blue light stimulation in the absence (control) or the presence of TeTX are shown on the right. m Summarized bar graphs show a significant enhancement of exocytosis of VAMP-2 laden vesicles in cultured NG2 cells after blue light stimulation. In TeTX, this increased exocytosis is completely abolished by the exocytosis blocker TeTX. Exocytosis events are analyzed from 5 and 6 NG2 cells for control and TeTX group, respectively. P value as indicated, two-tailed paired t test, n.s. indicates not significant. Data are presented as mean values ± SEM and error bar represents SEM.
Article Snippet: The primary antibodies included: rabbit antibody to NG2 (1:250, Millipore AB5320), Goat antibody to Pdgfrα (1:300, R&D Systems AF1062), rabbit antibody to GFAP (1:1500, Abcam ab7260), chicken antibody to GFP (1:500, Abcam ab13970), mouse antibody to Olig2 (1:500, Millipore MABN50), mouse antibody to CC1 (1:500, Millipore OP80), mouse antibody to cFos (1:1000, Abcam ab208942), mouse antibody to NeuN (1:500, Abcam ab104224), rabbit antibody to CCK-8 (1:1000, Sigma C2581), rabbit antibody to NPY (1:1000, Cell Signaling 11976 S), mouse antibody to PV (1:2000, Sigma P3088), rat antibody to SST (1:500, Millipore MAB354), guinea pig antibody to vGluT2 (1:200, Synaptic Systems 135404), rabbit antibody to VAMP-2 (1:1000, Alomone labs ANR-007), mouse antibody to Gephyrin (1:500, Synaptic Systems 147011), rabbit antibody to CaMKII (1:200, Abcam ab52476).
Techniques: Cell Culture, Transfection, Two Tailed Test, Fluorescence, Inhibition, Cotransfection, Purification, Expressing, High Performance Liquid Chromatography, Isolation, Concentration Assay, RNA Sequencing Assay, Plasmid Preparation
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![a Cartoon images illustrating the sniffer-patch method in co-cultured NG2 cells and HEK-293T cells. Scale bar, 20 μm. b Representative image showing primary cultured NG2 cells (in green) from Pdgfrα-creER TM ; ChR2-eYFP newborn mice brain with GABA A Rs-mCherry transfected HEK-293T cell (in red). Scale bar, 20 μm. c The representative trace shows an example of ChR2-evoked photocurrents by 100 ms blue light stimulation in a cultured NG2 cell. Bar graph shows −365.3 ± 25.1 pA peak photocurrent induced by blue light stimulation in cultured NG2 cells, n = 10 cells. d , e Representative traces ( d ) and summarized bar graph ( e ) show that 25 μM GABA application induces a distinct GABA A R-mediated current that is significantly abolished by GABA A R antagonist bicuculline in transfected HEK-293T cells. GABA A R-mediated currents: control: −526.2 ± 67.2 pA, n = 6 cells; in the presence of bicuculline: −32.9 ± 10.7 pA, n = 6 cells, two-tailed paired t test, P = 0.0005. f Representative images show robust [Ca 2+ ] i elevations in cultured NG2 cells loaded with the calcium indicator Rhod2-AM (5 µM) after 15 Hz 60 s blue light stimulation. Bar graph summary below shows the time course of blue light stimulation-induced [Ca 2+ ] i increase in cultured NG2 cells. n = 53 cells from 3 mice. Data are normalized by the mean fluorescence intensity obtained during the control period (0–30 s before the stimulation triggers) for each cell. g Representative traces and summarized bar graph show a significant increase of GABA A R-mediated tonic current in sniffer-patched transfected HEK-293T cell in the presence of blue light stimulation of cultured NG2 cells (middle panel) compared with its basal tonic inhibition in the absence of blue light stimulation (upper panel) obtained from a Pdgfrα-creER TM ;ChR2 mouse. This increased tonic GABA current is abolished by the cotransfection with exocytosis blocker TeTX in the presence of NG2 cells photoactivation (lower panel). Basal tonic GABA currents in transfected HEK cells without blue light stimuli: 6.57 ± 0.82 pA, n = 10 cells; tonic GABA currents in the presence of blue light stimuli: 28.27 ± 3.61 pA, n = 15 cells; tonic GABA currents in the presence of TeTX and blue light stimuli: 12.38 ± 2.33 pA, n = 12 cells. ANOVA Tukey–Kramer Multiple Comparisons, *** indicates P < 0.001. h Bar graph summary showing increased GABA concentrations in purified ChR2-expressing NG2 cells after blue light stimulation compared with its control with high-performance liquid chromatography (HPLC) analysis. n = 4 and 5 independent experiments for ChR2 + -NG2 cells and ChR2 - -NG2 cells, respectively. P values as indicated, two-tailed paired t test. i GABA levels in synaptosomes isolated from purified NG2 cells and GAD67-GFP interneurons with HPLC. Bar graph showing an average GABA concentration of 5.76 ± 1.04 ng/mg synaptosomes in NG2 glia (right panel). n = 5 tested samples. j The bar graph summary shows GABA contents in synaptosomes in both purified NG2 cells and GAD67+ interneurons compared with hippocampal tissue. n = 11 tested samples for purified primary cultured NG2 cells, n = 2 and 3 tested samples for GAD67 interneurons and hippocampi from 4 GAD67-GFP and 3 C57BL/6 mice, respectively. k The pie graph shows a component percentage of vesicle-associated membrane protein (VAMP)-encoded genes through RNA-sequencing data analysis of isolated NG2 glia in adult brain by FACS at postnatal 3–4 weeks. The representative image below shows a precise colocalization of transfected VAMP2-pHuji plasmid (in red) with anti-VAMP2 antibody (in green) in primary cultured NG2 cells. Scale bar, 20 μm. l Representative TIRFM images show the total VAMP2-pHuji laden vesicular fusion events at 0, 2.5, 5, 7.5, and 10 s with the image size of 10 × 10 μm 2 before (upper panel) and after (lower panel) blue light stimulation (10 Hz, 60 s) in the absence (control panels) or the presence of TeTX (TeTX panels), respectively. The average cumulative graphs of fusion events during a 10 s time window before and after blue light stimulation in the absence (control) or the presence of TeTX are shown on the right. m Summarized bar graphs show a significant enhancement of exocytosis of <t>VAMP-2</t> laden vesicles in cultured NG2 cells after blue light stimulation. In TeTX, this increased exocytosis is completely abolished by the exocytosis blocker TeTX. Exocytosis events are analyzed from 5 and 6 NG2 cells for control and TeTX group, respectively. P value as indicated, two-tailed paired t test, n.s. indicates not significant. Data are presented as mean values ± SEM and error bar represents SEM.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4468/pmc08484468/pmc08484468__41467_2021_25956_Fig5_HTML.jpg)
